Ricin and Shiga toxin share a unique mode of action, causing irreversible damage to host ribosomal RNA. Our understanding of how these bioterror agents cause tissue damage is incomplete and no medications exist to prevent or treat intoxication with these agents. Shiga toxin- or ricin-mediated ribosomal damage results in inhibition of protein synthesis as well as profound changes in host cell signal transduction. This phenomenon, called the "ribotoxic stress response", is poorly understood. This proposal is focused on understanding: 1) what initiates the ribotoxic stress response; 2) which signal transduction pathways are involved; and 3) what are the downstream effects that may contribute to disease pathogenesis. Our preliminary data indicate that ricin and Shiga toxin activate host stress activated protein kinase (SAPK) and mitogen activated protein kinase (MAPK) pathways. We hypothesize that activation of these pathways contributes to intestinal and other tissue damage observed following intoxication with these agents. Effects of ricin and Shiga toxins on these pathways will be studied using cultured intestinal epithelial cells and an infant rabbit model of oral intoxication. The mechanism(s) by which these signal transduction pathways are stimulated will be assessed using Shiga toxin mutants and isolated Shiga toxin subunits. Direct effects of ricin and Shiga toxin on host cell signal transduction will be studied using phosphorylation-specific antibodies and immunoprecipitation kinase assays. Transfection of dominant negative constructs, RNA interference, and specific chemical inhibitors of signal transduction pathways will be used to dissect the mechanisms by which ricin and Shiga toxin alter cell physiology. Our work has revealed that oral administration of purified Shiga toxin 2 induces gut damage. In the infant rabbit model, SAPKinase-blocking compounds will be tested for their effect on preventing the gut damage that occurs following oral administration of Shiga toxin or ricin. This work will provide new insight into the ribotoxic stress response. Understanding how Shiga toxin and ricin cause damage to host cells should reveal potential therapeutic targets. These findings will be relevant to either accidental or intentional intoxication with ricin or Shiga toxin, and may prove generalizable to other host target organs affected by these toxins.